Composition and method for modifying the fatty acid composition of cell membranes of organs and tissues

ABSTRACT

The present invention relates to a composition and method for rapidly modifying the fatty acid composition of cell membranes of organs and tissues, in particular to increase the amount of omega-3 fatty acids of cell membranes of organs and tissues by parenterally administering to the human or animal body a supply of fatty acids in the form of an isotonic lipid emulsion comprising fatty acid triglycerides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is the National Phase filing in the United States ofInternational Application Number PCT/EP02/08121, filed Jul. 20, 2002,and claims priority of European Patent Application No. 01117991.8 filedJul. 25, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a composition and method for rapidlymodifying the fatty acid composition of cell membranes in organs andtissues, in particular to increase the amount of omega-3 fatty acids incell membranes of organs and tissues by parenterally administering tothe human or animal body an appropriate supply of fatty acids in theform of an isotonic lipid emulsion comprising selected fatty acidtriglycerides.

(2) Description of Related Art Including Information Disclosed Under 37C.F.R. §§ 1.97 and 1.98.

Lipid emulsions have been known as an essential component of parenteralnutrition and are now being considered for other uses. Lipid emulsionsfor parenteral nutrition serve to supply the body with fats in anintravenously acceptable dosage form in cases where normal (oral)nutrition is impossible, comprised or medically contraindicated or whenit is necessary to promptly modify the fatty acid pattern of the cells.The lipid emulsions currently available are prepared from vegetable oils(e.g., safflower or soybean oils). In some cases they also containmedium-chain triglycerides (MCT) and/or oils of marine origin (fishoils).

Long-chain triglycerides of vegetable or marine origin serve as anenergy source and, when containing polyunsaturated fatty acids (“PUFA”),as suppliers of essential fatty acids. The classification of suchpolyunsaturated fatty acids into omega-6 (ω-6); in the art sometimesdesignated “n-6” PUFA) or omega-3 (ω-3, sometimes designated “n-3” PUFA)series is based on chemical structural features, more precisely, on thedistance of the first unsaturated bond from the methyl end (omega end)of the fatty acid molecule. In the present description, for instance,“omega-3” has preferably been used.

The vegetable oils, e.g., of soybean and safflower, are characterized bya high content of polyunsaturated fatty acids of the omega-6 series(predominantly Linoleic acid, 18:2 omega-6) whereas their content ofomega-3 fatty acids (almost exclusively in the form of α-linolenic acid,18:3 omega-3 is low.

Fish oils (“FO”) obtained from cold-water fish are characterized by ahigh content of polyunsaturated fatty acids of the omega-3 series(predominantly cis-5,8,11,14,17-eicosapentaenoic acid, “EPA,” 20:5omega-3, docosapentaenoic acid, “DPA,” 22:5 omega-3 andcis-4,7,10,13,16,19-docosahexaenoic acid, “DHA,” 22:6 omega-3) whereastheir content of omega-6 fatty acids is low.

The medium-chain triglycerides (“MCT”) administered with the lipidemulsions serve mainly as a source of energy. Medium-chain triglyceridescontain saturated fatty acids and hence contain neither the omega-6 noromega-3 essential fatty acids. Because of their fast hydrolysis as wellas other properties (enhancing particle binding to cells), MCT may haveinteresting influences on the metabolism of emulsion particles.

The human body is itself incapable of producing the vital,polyunsaturated long-chain fatty acids of the omega-6 or omega-3 series,i.e., they have to be administered orally, enterally or parenterally.The body is only able to synthesize longer-chain unsaturated fatty acidsfrom shorter-chain ones. Both series compete for the same enzymaticsystem of elongation-desaturation. The formation of omega-6 fatty acidsfrom precursors of the omega-3 series or vice versa is impossible,however.

In order that the exogenous free fatty acids are made available to thebody, they must either be released hydrolytically from the infusedtriglycerides by means of the enzyme lipoprotein lipase (LPL) or betaken up together with emulsion particles or their remnants directlyinto the cells. This initial step of lipid hydrolysis has long beenconsidered the rate-determining step of lipid metabolism. Thislimitation arises from the relatively limited activity of lipoproteinlipase in cleaving triglycerides. Thus, the maximum metabolizing ratefor vegetable oil emulsions is about 3.8 g of lipid/kg body weight perday (Hallberg et al., Acta Physiol. Scand. (1965) Vol. 65, Suppl. 254,pp. 2-23).

During triglyceride infusion, it is desirable to achieve triglycerideserum concentrations which remain as low as possible, e.g.,corresponding to a low load of the reticulo-endothelial system (RES) byexogenous lipids.

Typically, post-operative and post-traumatic conditions as well assevere septic episodes are characterized by a substantial stimulation ofthe immune system. The immune response involves the release of cytokines(e.g., tumor necrosis factor and interleukins) which, at high levels,may cause severe tissue damage. In addition, high cytokineconcentrations also impair hydrolysis of circulating triglycerides byLPL.

In such clinical conditions, it is of particular importance to useexogenous triglycerides which are rapidly hydrolyzed and eliminated (toavoid excessive increases of plasma triglyceride concentration) andwhich supply fatty acids (e.g., omega-3 fatty acids) capable of reducingcytokine production as well as cytokine toxicity on tissues. This effectis obtained when fatty acids are cleaved from the triglyceride moleculesand incorporated (in free form or as components of phospholipids) incell membranes where they influence membrane structure (and function)and serve as secondary messengers and precursors of eicosanoids. Thus,it is desirable that this process take place as quickly as possible.

Triglycerides typical of fish oils are hydrolyzed much more slowly thantriglycerides from vegetable oils (e.g., soybean oil) which arethemselves hydrolyzed more slowly than medium-chain triglycerides.Addition of a fish oil emulsion to a long-chain triglyceride emulsioncan even inhibit hydrolysis of long-chain triglycerides (e.g., fromsoybean oil) by LPL.

Description of Related Art Including Information Disclosed Under 37 CFR1.97 and 1.98

European published patent application EP-A-0311091 discloses a lipidemulsion comprising medium chain triglycerides and a high amount of fishoil for parenteral nutrition.

International published patent application WO-A-90/08544 discloses fatemulsions comprising, as a source for omega-3 fatty acids, fish oil and0 to 90%, based on the total lipid, of medium chain triglycerides andtheir intraperitoneal application for the treatment of septic affectionof the abdominal cavity.

International published patent application WO-A-97/19683 discloses lipidemulsions comprising medium chain triglycerides, vegetable oils and fishoils for parenteral nutrition. WO-A-97/19683 also discloses the utilityof said lipid emulsions for treating post-surgery, post-trauma, sepsis,inflammatory or wasting diseases, increased risk of vascular thrombosisand severe cardiac arrhythmia.

European published patent application EP-A-0687418 provides a lipidemulsion to limit the injury response in patients suffering from trauma,burns and/or sepsis, which lipid emulsion could be administeredenterally or parenterally.

Simoens, C. et al. in Clinical Nutrition (1995), 14, 177-185 disclosesthe effects of the fatty acid composition in various tissues of fourdifferent vegetable-oil containing lipid emulsions. Sato, M. et al. inJournal of Parenteral and Enteral Nutrition (1994), 18, 112-118,discloses the hydrolysis of mixed lipid emulsions containing mediumchain and long chain triglycerides with lipoprotein lipase in aplasma-like medium.

German published patent application DE-A-3721137 discloses the utilityof a lipid emulsion comprising fish oil alone or fish oil in combinationwith vegetable oil and optionally medium chain triglycerides toparenteral nutrition and the reduction of the growth of tumors.

German published patent application DE-A-3409793 describes a lipidemulsion for infusion, which emulsion comprises fatty acids containingfrom 20 to 22 carbon atoms, esters thereof, or a mixture of 2 or more ofsuch fatty acids or esters, as well as a vegetable oil, an emulsifier,and water. The fatty acids are fatty acids from esters of marine origin(fish oils), in particular, omega-3 fatty acids. Said vegetable oils arepurified soybean and/or safflower oils.

The plasma clearance and tissue targeting of different intravenous lipidemulsions (fish oil; MCT/vegetable oil/fish oil; vegetable oil andmCT/vegetable oil) was compared in a mouse model by Treskova, E. et al.in Journal of Parenteral and Enteral Nutrition (2000), 23, 253-257.Billman, G. E. et al. in Circulation (1999), 99, 2452-2457 convincinglydemonstrate that omega-3 fatty acids administered intravenously as theirpure free fatty acid can prevent ischemia-induced ventriculararrhythmias in dogs.

From the above it can be derived that the major well-recognized roles ofomega-3-polyunsaturated fatty acids are:

to decrease inflammatory and thrombotic reactions,

-   -   (i) to reduce cell reactivity to different stimuli (for example,        to reduce cardiac arrhythmias, namely during myocardial infarct        or ischemia, and to decrease cachexia in response to mediators        such as TNFα, in conditions of cancer and inflammation),    -   (ii) to improve tissue micro-perfusion (e.g., during shock or        after ischemia-reperfusion),    -   (iii) to improve intracellular antioxidant status (in spite of        the well known sensitivity of PUFAs to peroxidation, which may        be controlled by adequate amounts of liposoluble antioxidants),        and    -   (iv) to limit intracellular fat accumulation.

In addition, omega-3 PUFAs are essential for the maturation of thecentral nervous system (CNS) and the retina in the fetus and prematurenewborns. However, the rate of omega-3 fatty acid enrichment followingoral supplementation substantially varies between different tissues andis particularly low in some regions of the brain and in the retina.

Although omega-3 fatty acids play essential functions at all thesedifferent levels, evolution of food intake in mankind is characterizedby an important decrease in the consumption of omega-3 fatty acids and arise of omega-6 fatty acid intake, especially in Western populations.

Still, benefits of omega-3 fatty acid supplementation have beenconfirmed in several clinical conditions with a strong correlation toomega-3 fatty acid concentration into cell membrane phospholipids.

BRIEF SUMMARY OF THE INVENTION

It has been an object of the present invention to provide a method andcomposition allowing for a very rapid and efficient uptake/enrichment ofomega-3 fatty acids into cell membranes of organs and tissues. Anefficient uptake of omega-3 fatty acids into cell membranes of organsand tissues means that only a fraction (or none) of the administeredomega-3 fatty acids or its precursors is oxidized and that most of them(or all) are incorporated into organs and tissues and namely cellmembranes. Enriching cell membrane and its phospholipids with omega-3long chain polyunsaturated fatty acids is, under certain circumstances,necessary to (at least partly) restore an adequate balance betweenomega-3 and omega-6 fatty acids. To achieve those effects is not onlyessential for patients in acute conditions but for all patients with anincreased need for omega-3 fatty acids in cell membrane phospholipidsand when the intravenous route offers advantages, such as in case ofpreterm infants and patients with cachectic (wasting) diseases and thosebeing unable to absorb large amounts of omega-3 fatty acids.

A first option would be the infusion of pure omega-3 PUFA. However,since unbound fatty acids have a high degree of cytotoxicity, the onlypossible way to infuse pure omega-3 PUFA is to bind them to albumin (asthey are in plasma). The infusion rate should be very slow to avoidmarked rises of plasma concentration. The presence of albumin wouldincrease the size of plasma compartment and also markedly increase thecosts.

A second option would be the infusion of a pure fish oil emulsion.However, pure fish oil emulsion particles have a very slow eliminationrate and the incorporation of omega-3 fatty acid into cell membranephospholipids obtained with pure fish oil emulsions is quite inefficientwhen considering their high content in omega-3 PUFA. This may beexplained by the fact that a substantial proportion is metabolized intoother pathways such as oxidation.

A third option would be the infusion of MLF 541, a lipid emulsion whichis known from International published patent application WO-A-97/19683.Said lipid emulsion comprises 50% MCT (“M”), vegetable oil (“L”) and 10%of fish oil (“F”), based on the total amount of lipids. It has beenfound, however, that this emulsion maintains the balance between omega-3and omega-6 fatty acid in membrane phospholipids, but does not induce aspecific enrichment of omega-3 fatty acids.

As is evident from the above, the present invention aims at parenteraladministration but is not directed to a balanced supply of fatty acidsfor the purpose of nutritional support/nutrition.

This and other objects of the invention have been solved by an isotoniclipid emulsion comprising medium chain triglycerides (MCT) and fish oil.The emulsions of the invention do not contain vegetable oils.

In particular the present invention relates to an isotonic lipidemulsion comprising

-   -   (i) about 60 to about 95% by weight of medium chain        triglycerides (MCT), and    -   (ii) about 5 to 40% by weight of fish oil,        based on the total amount of lipids in the emulsion, under the        proviso that the emulsion does not contain vegetable oils.

It has surprisingly been found that the omega-3 fatty acid content ofcell membranes of certain key organs and tissues can significantly beincreased by administering the lipid emulsion of the present invention.Compared to a similar emulsion comprising fish oil as the onlytriglyceride source or a combination of fish oil, vegetable oil andmedium-chain triglyceride the above lipid emulsion of the presentinvention is much more efficient although less fish oil is employedcompared to the pure fish oil emulsion and although the emulsion isdevoid of any vegetable oil. It has further been found that the ratio ofMCT to fish oil has a significant impact on the effects achieved.

According to the invention the medium-chain triglycerides contain fattyacids having from 6 to 14 carbon atoms. Preferably, at least 90% byweight of the MCTs are triglycerides of caprylic acid (C₈) and capricacid (C₁₀). The fraction of medium-chain triglycerides in the lipidemulsion of the invention is preferably from 70% to 90%, more preferablyfrom 78% to 85% by weight, based on the total lipid content of the lipidemulsion.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

Fish oils are known to contain eicosapentaenoic acid (EPA, 20:5 omega-3)and docosahexaenoic acid (DHA, 22:6 omega-3) incorporated intriglycerides which, being so-called highly unsaturated omega-3 fattyacids, are essential building blocks which have to be supplied to thebody and which are biologically important, for example, as precursors ofeicosanoids and as structural elements of membrane lipids. These acidsare further attributed antithrombotic and lipid-lowering actions. Sincetheir isolation from natural products and their chemical synthesis isexpensive, fish oils, being relatively inexpensive, are the suppliers ofchoice for such essential fatty acids. As used in the invention, theterm “fish oils” is intended to comprise natural fish oils, processedfish oils, highly purified fish oil concentrates or (re-) esterified(synthetic fish oils. Processed fish oils are described in Europeanpublished patent application EP-A-0298293, the disclosure of which isincorporated herein by reference.

Suitable exemplary fish oils are oils which are obtained from cold-waterfish on a technically significant scale. Typical cold-water fish areselected from salmon, sardine, mackerel, herring, anchovy, smelt andswordfish. Likewise the term fish oil is meant to comprise oils whichare synthetically obtainable by (re-) esterification of omega-3 fattyacids as obtained from fish oil of the above cold water fish byhydrolysis of the triglycerides and subsequent purification andconcentration of the resultant omega-3 fatty acids with glycerol. Fishoils generally contain glycerides of fatty acids having chain lengths offrom 12 to 22 carbon atoms. Particularly preferred are highly purifiedfish oil concentrates which are obtained, for instance, from sardine,salmon, herring and/or mackerel oils. They have an eicosapentaenoic acidcontent of from 20 to 40%, preferably at least 25%, based on the fattyacid methyl esters of the fish oil concentrate as determined by gaschromatography (percent by area). Furthermore, they have adocosahexaenoic acid content of from 10 to 20%, preferably at least 12%,based on the fatty acid methyl esters of the fish oil concentrate asdetermined by gas chromatography (percent by area). In case of the fishoils which are synthetically obtainable by the re-esterification of theomega-3 fatty acids the total concentration of eicosapentaenoic anddocosahexaenoic acid can be raised to at least 45%, on the basis of thetotal amount of triglycerides. U.S. Pat. No. 6,159,523 discloses amethod for making fish oil concentrates. Generally, the amount of thepolyunsaturated fatty acids of the omega-6 series (e.g., linoleic acid,18:2 omega-6) in natural fish oils is low and generally less than 10%,preferably less than 5%.

It is particularly preferred to use a fish oil rich in EPA wheninflammatory processes are to be influenced. Fish oil rich in DHA isparticularly preferred in pediatric patients in the case of omega-3fatty acid deficiency to influence growth and maturation of the centralnervous system.

Preferably, the content of fish oil in the emulsion according to theinvention is from about 5% to about 30%, more preferably from about 15%to about 22% by weight, based on the total lipid content of the lipidemulsion.

The total lipid content (MCT+fish oil) of the lipid emulsion is fromabout 5% to about 30%, preferably from about 10% to about 25% by weight,based on the total weight of the aqueous lipid emulsion.

Throughout the description the indication of “% by weight” inconjunction with the lipid concentration in the emulsion caninterchangeably be used with “g lipid per 100 ml emulsion.”

In addition to distilled water the isotonic lipid emulsion may containconventional auxiliary agents and/or additives, such as emulsifiers,emulsifying said (co-emulsifiers), stabilizers, antioxidants, andisotonizing additives.

As emulsifiers, physiologically acceptable emulsifiers (surfactants) areused, such as phospholipids of animal or vegetable origin. Particularlypreferred are purified lecithins, especially soybean lecithin, egglecithin, or fractions thereof, or the corresponding phosphatides. Theemulsifier content may vary from about 0.02 to about 2.5% by weight,preferably from about 0.6% to about 1.5% and most preferably about 1.2%by weight, based on the total emulsion.

Further, alkali metal salts of long-chain, C₁₆ to C₂₀ fatty acids may beused as emulsifying aids (co-emulsifiers). Especially preferred aretheir sodium salts. The co-emulsifiers are employed in concentrations offrom about 0.005% to about 0.1%, preferably about 0.02% to about 0.04%by weight, based on the total emulsion. Further, cholesterol, acholesterol ester alone or in combination with other co-emulsifiers maybe employed in a concentration of from about 0.005% to about 0.1%,preferably from about 0.02% to about 0.04% by weight, based on theemulsion.

The lipid emulsion according to the invention may contain an effectiveamount of an antioxidant, such as vitamin E, in particular α-tocopherol(which is the most active isomer of vitamin E in man) as well as β- andγ-tocopherol, and/or ascorbyl palmitate as antioxidants and thus forprotection from peroxide formation. The total amount of said antioxidantmay be up to 5000 mg. In a preferred embodiment the total amount of saidantioxidant may vary between from about 10 mg to about 2000 mg, morepreferably from about 25 mg to about 1000 mg, most preferably from about100 mg to 500 mg, based on 100 g of lipid.

For stabilization and isotonization, the emulsion according to theinvention may contain from about 2% to about 5% by weight, based on theemulsion, of a stabilizing or isotonizing additive, for example, apolyhydric alcohol. Glycerol, sorbitol, xylitol or glucose arepreferred, glycerol being particularly preferred.

The isotonic lipid emulsion is supplied to the subject in thepharmaceutically effective amount necessary for the respectivetreatment. The administration can be continuous or in the form of one orseveral doses per day. By pharmaceutically effective amount there ismeant about 0.1 to about 1.0 g TG (triglyceride) per one kg bodyweightper day, preferably about 0.2 g to about 0.5 g TG per one kg bodyweightper day, most preferably about 0.25 g to about 0.33 g TG per one kgbodyweight per day.

The key tissues are selected from endothelium, white blood cells,platelets and immune cells. The organs are selected from heart, brain,kidney, lung, liver and fat.

The isotonic lipid emulsion and method of the present invention can beused for the treatment of patients with a global omega-3 fatty aciddeficiency or a relative deficiency in cell membranes of certain organsand tissues. In particular, the emulsion and method of the presentinvention is of great value for the treatment of surgical orpercutaneous re-vascularization, such as in coronary or other“peripheral” arteries, myocardial ischemia or infarction, unstableangina, transient cerebral ischemia or stroke, inflammation,auto-immune, and thrombotic diseases, such as venous or arterialdiseases, organ transplantation (infusion in both donors andrecipients), angiographic procedures, hemodialysis, preterm infants,acute phase reactions, acute respiratory distress syndrome, intestinalischemia, cardiovascular complications of diabetes mellitus, severeburns, severe cachexia, Raynaud's disease, and omega-3 fatty aciddeficiency in cell membranes.

The isotonic lipid emulsions of the invention can be utilized for themanufacture of medicaments/pharmaceutical compositions for the treatmentof the above-mentioned diseases.

The lipid emulsions according to the invention are invariablyoil-in-water (o/w) emulsions in which the outer continuous phaseconsists of distilled water purified for parenteral purposes. Suchoil-in-water (o/w) emulsion is obtained by mixing the MCT and fish oilfollowed by emulsification and sterilization. The pH value of the lipidemulsion is adjusted to a physiologically acceptable value, preferablyto a pH of from about 6.0 to about 9.0, more preferably from about 6.5to about 8.5. As is evident from the described method of preparation,the emulsion of the invention is an emulsion of a mixture of thetriglycerides. The auxiliary agents and additives can be added to theMCT/fish oil mixture prior to emulsification or they can be added to theemulsion prior to sterilization.

The isotonic lipid emulsions according to the invention can be preparedby known standard procedures with inertization. The usual approach isfirst to mix the lipids, emulsifier and other auxiliary agents andadditives and then to fill up with water with dispersing. The water mayoptionally contain additional water-soluble components (e.g., glycerol).The emulsion thus obtained still contains lipid particles having adiameter of about 10 μm. The average droplet size of the emulsion mustthen be reduced further by additional homogenization, e.g., using ahigh-pressure homogenizer. For parenteral application, medium lipiddroplet sizes of less than about 1.0 μm, in particular less than about0.8 μm, most preferably less than about 0.5 μm are preferred. Preferablythe lipid emulsions of the invention are intravenously injectable. Thusthe present invention also relates to a pharmaceutical compositioncomprising the isotonic lipid emulsion as described above, preferablyfor injection into the human or animal body.

The lipid emulsions according to the invention can be used forparenteral administration in patients with impaired tissue or organperfusion or increased risk of severe cardiac arrhythmia (e.g.,ventricular fibrillation) or vascular thrombosis or severe cardiacarrhythmia or exaggerated inflammatory responses, acute respiratorydistress syndrome, or during dialysis in patients treated withhemodialysis or to promptly raise the omega-3 fatty acid content in thebrain and retina of preterm infants. The invention can be used inpatients in pre-operative conditions prior to a re-vascularizationprocedure or in post-operative conditions or with inflammatory diseases;further, in severe or persistent post-aggression metabolic responsefollowing operations, such as abdominal operations or organtransplantations, and multiple trauma, inflammatory diseases, burns,infections, impending or manifest sepsis, wasting diseases.

The invention will be illustrated further by the following examples butshould not be construed to be limited to these.

EXAMPLES

The following Table shows the fatty acid composition (approx. %) ofvarious oils used in the lipid emulsions of the following examples:

TABLE Fatty acid MCT oil¹⁾ Soybean oil²⁾ Safflower oil³⁾ Fish oil⁴⁾ 6:0<2 — — — 8:0 64 — — — 10:0  34 — — — 12:0  <3 — — <1 14:0  <1 — — 516:0  — 11 — 10 16:1  — — — 7 16:2  — — — 1 16:3  — — — 1 16:4  — — — 318:0  — 4 3 1 18:1  — 22 14 10 18:2 omega-6 — 55 75 2 18:3 omega-3 — 8<1 1 18:4 omega-3 — — — 4 20:0  — <1 <1 — 20:1  — <1 <1 2 20:4 omega-6 —— — 2 20:5 omega-3 — — — 28 22:1  — — — 1 22:4  — — — <1 22:5  — — — 322:6 omega-3 — — — 13 Σ omega-6 — 55 75 4 Σ omega-3 — 8 <1 46 omega-6 to— 7:1 >75:1 1:12 omega-3 ratio ¹⁾medium chain triglycerides, e.g.,Captex 355, commercial product of Karishamns. ²⁾soybean oil, e.g.,“Sojaöl,” commercial product of Croda. ³⁾safflower oil, e.g.,“Safloröl,” commercial product of Gustav Heess. ⁴⁾highly purified fishoil, e.g., Sanomega S28GA, commercial product of Nippon Oil and Fats.

The lipid emulsions in the examples comprising MCT, fish oil andemulsifier (about 1.2 g per 100 mL emulsion fractionated phospholipidsfrom chicken egg yolk) and optionally the vegetable oil are made usingstandard industrial methods for the production of therapeutic emulsionsin water by dispersing the ingredients by means of an Ultra-Turrax andfilled up with the aqueous component with stirring. The pH value isadjusted to pH 8.0 to 9.0 using an aqueous sodium hydroxide solutionand/or sodium oleate. Subsequent homogenization is performed in ahigh-pressure homogenizer at 400 kg/cm². After dispensing in glassbottles of appropriate grade, heat sterilization is performed by knownmethods. The emulsions tested had a total lipid content of 20 g per 100mL emulsion, based on the total emulsion, corresponding to about 20% byweight. The mean particle size of the emulsion particles is about 300 nmand may vary ±20%. All emulsions have about the same emulsion particlediameter. The emulsions MLF 541, fish oil 100%, and MCT/fish oil 5:5 arenot according to the invention and for comparison only.

Materials and Methods

Prior to addition to cell culture, lipid preparations were incubated inplasma for 45 min at 25° C., at a final concentration of 250 mg TG/dL(TG=triglyceride) and emulsion triglyceride-rich particles (TGRP) wereseparated by short term ultra-centrifugation (30 min; 20,000 g). Inaddition, HUVEC (human umbilical vein endothelial cells) were incubatedwith bovine lipoprotein lipase (8 μg/mL in DMEM) for 30 min at roomtemperature and then washed (once with 3% BSA-containing DMEM (Gibco)and twice with DMEM).

TGRP were then added to cell culture medium (containing 3% BSA) at afinal concentration of 500 μg TG/mL (50 mg/dL). Incubations wereperformed at 37° C. for 4 h. Endothelial cells were washed once with0.02% BSA-containing DMEM and twice with DMEM prior to being detached bytrypsin-EDTA solution.

Fatty acid pattern was determined by gas liquid chromatography in lipidcomponents (TG, CE [=cholesteryl esters], PL) previously separated bythin layer chromatography (Lepage G. and Roy C. C., J. Lipid Res.(1986), 27:114-120).

RESULTS Preliminary Experiments

In a first series of experiments, we tested the reproducibility of ourin vitro model with respect to the endothelial cell growth in culture.Indeed, it is essential to develop conditions guaranteeing a very stablean confluent mono-layer (and to avoid over-confluency) in dishes beforeincubations, and to test variations of fatty acid composition duringendothelial cell growth. This series of experiments has demonstratedthat HUVEC have to be cultured in medium containing 10% serum for 5 daysto reach confluency and that growth can then be inhibited by a 2-dayculture in a serum-deficient medium, which guarantees a stable fattyacid composition.

A second set of experiments was designed to optimize incubation time forreaching significant incorporation of omega-3 PUFA. These experimentshave shown that, considering a triglyceride concentration of 50 mg/dL(which is lower than the average rise of plasma triglycerides duringlipid infusion) in the medium, incubation for 4 h leads to substantialand nearly maximum changes in endothelial cellphospholipids. Indeed,incubations for 20 h did not show major additional modificationscompared to 4 h, but led to an increased intracellular triglyceridecontent and partly induced cell death.

After a 4 h incubation with TGRP, lipid-containing medium was removedand replaced with a fat-free medium. Cells were then cultured foradditional 2 h or 20 h to evaluate the stability of fatty acid patternin endothelial cells. The total omega-3 fatty acid content in cellphospholipids remained unchanged after 2 h and 20 h. However, slightchanges of omega-3 PUFA distribution were found after 20 h, which mainlyconsisted of an increased DPA (docosapentaenoic acid) content (at theexpense of EPA). These modifications may derive from elongation anddesaturation pathways taking place into cell membrane phospholipids.

Influence of the Emulsion Triglycerides on the Omega-3 PolyunsaturatedFatty Acid Incorporation in Cell Phospholipids

Cells were cultured as described above and were then incubated for 4 hin the absence (control cells) or in the presence of the twoexperimental TGRP, namely MCT/fish oil 8:2 and MCT/fish oil 5:5 bycomparison to MLF 541 (according to International published patentapplication WO-A-9719683, Lipoplus®, B. Braun Melsungen AG, Germany) anda pure fish oil (FO 100%) emulsion.

TABLE 1 Fatty acid composition of endothelial cell phospholipids after 4h incubation with TGRP (mean values ± SD, n = 4) Fatty acid pattern(weight %) EPA (C20:5 ω-3) Δ DPAC22:5 ω-3) Δ DHA (C22:6 ω-3) Δ Controlcells 0.65 ± 0.06 3.84 ± 0.29 2.54 ± 0.28 MLF 541 2.09 ± 0.15 1.44 4.35± 0.15 0.51 2.85 ± 0.16 0.31 FO 100% 5.39 ± 0.29 4.74 4.73 ± 0.31 0.893.44 ± 0.21 0.90 MCT/FO 8:2 4.55 ± 0.31 3.90 5.35 ± 0.33 1.51 3.45 ±0.23 0.91 MCT/FO 5:5 5.83 ± 0.04 5.18 5.50 ± 0.35 1.66 3.55 ± 0.22 1.01

The rise of EPA in cell phospholipids induced by incubation withMCT/fish oil 8:2 represents respectively 75 and 82% of the augmentationobserved with MCT/fish oil 5:5 and pure fish oil. MCT/fish oil 8:2 wasat least as efficient as all other preparations to induce an incrementof DPA (docosapentaenoic acid, C22:5 omega-3) and DHA.

TABLE 2 omega-3 PUFA increase in endothelial celiphospholipids (Δ)relative to the omega-3 fatty acid content in lipid preparations(expressed as weight-% fatty acid enrichment/fish oil proportion) Lipidemulsion EPA (C20:5 ω-3) DPA (C22:5 ω-3) DHA (C22:6 ω-3) MLF 541 14.45.1 3.1 OF 100% 4.7 0.9 0.9 MCT/OF 8:2 19.5 7.6 4.5 MCT/OF 5:5 10.5 3.32.02

Taking into account the respective fish oil content in the differenttested preparations, MCT/fish oil 8:2 appeared to induce a moreefficient incorporation of EPA, DPA and DHA in cell phospholipids thanall other preparations.

MLF 541 also showed a remarkable efficacy in enriching endothelial cellswith omega-3 fatty acid while pure fish oil and to a lesser extentMCT/fish oil 5:5 showed a relatively poor efficacy by comparison totheir higher fish oil content.

TABLE 3 omega-3/omega-6 fatty acid ratio in endothelial cell membranephospholipids after treatment with MLF 541, MCT/fish oil 8:2 or purefish oil (in wt.-%) ω-3/ω-6 Lipid emulsion Σ ω-3 fatty acids Σ ω-6 fattyacids ratio None/Control cells 6.8 11.6 0.581 MLF 541 9.4 18.5 0.506 OF100% 13.8 12.0 1.149 MCT/OF 8:2 13.3 12.3 1.083

With respect to the omega-3/omega-6 fatty acid ratio in cellphospholipids, the MCT/fish oil 8:2 and FO 100% induce a two-foldincrease in the balance while MLF 541 affects both omega-3 and omega-6fatty acid contents and therefore does not substantially modify theratio.

The foregoing description has been directed to particular embodiments ofthe invention in accordance with requirements of the Patent Statutes forthe purposes of illustration and explanation. It will be apparent,however, to those skilled in this art, that many modifications, changesand variations in the claimed lipid emulsions, methods of treatment andmethods of administering the lipid emulsions set forth will be possiblewithout departing from the scope and spirit of the claimed invention. Itis intended that the following claims be interpreted to embrace all suchmodifications and changes.

1. An isotonic lipid-in-water emulsion free of long-chain vegetableoils, said emulsion comprising I. 78 to 95% by weight of medium chaintriglycerides (MCT), and II. 5 to 22% by weight of fish oil, based onthe total amount by weight of MCT and fish oil lipids in the emulsion,wherein the fish oil has an eicosapentaenoic acid content from 20 to 40percent based on the fatty acid methyl ester of the fish oil.
 2. Theisotonic lipid emulsion of claim 1, wherein the medium chaintriglyceride fatty acids contain between 6 and 14 carbon atoms.
 3. Theisotonic lipid emulsion of claim 2, wherein the medium chaintriglycerides are comprised of at least 90% of triglycerides of caprylicacid (C₈) and capric acid (C₁₀).
 4. The isotonic lipid emulsion of claim1, wherein the fish oil is selected from sardine, salmon, herring,mackerel and/or other cold water fish oils or fish oils syntheticallyobtainable by reesterification of glycerol with omega-3 fatty acidobtained by hydrolysis of cold water fish oils.
 5. The isotonic lipidemulsion of claim 1, wherein the fish oil comprises triglycerides whichcontain at least 25% by weight of eicosapentaenoic acid, based on thetotal weight of fatty acids of the fish oil.
 6. The isotonic lipidemulsion of claim 1, wherein the fish oil comprises triglycerides whichcontain at least 12% by weight of docosahexaenoic acid based on thetotal weight of fatty acids of the fish oil.
 7. The isotoniclipid-in-water emulsion of claim 1, wherein the total lipid content is 5to 30% by weight, based on the total volume of the lipid emulsion. 8.The isotonic lipid emulsion of claim 1, further comprising at least oneauxiliary agent or additive selected from the group consisting ofemulsifiers, emulsifying acids (co-emulsifiers), stabilizers,antioxidants and isotonizing additives.
 9. A method for increasing theomega-3 fatty acid content of cell membranes of key organs and tissuesin a human or animal body by administering to the subject atherapeutically effective amount of an isotonic lipid-in-water emulsionfree of long-chain-vegetable oils, said emulsion comprising I. 78 to 95%by weight of medium chain triglycerides (MCT), and II. 5 to 22% byweight of fish oil, based on the total amount by weight of MCT and fishoil lipids in the emulsion, wherein the fish oil has an eicosapentaenoicacid content from 20 to 40 percent based on the fatty acid methyl esterof the fish oil.
 10. The method of claim 9, wherein the medium chaintriglyceride fatty acids contain between 6 and 14 carbon atoms.
 11. Themethod of claim 9, wherein the medium chain triglycerides are comprisedof at least 90% of triglycerides of caprylic acid (C₈) and capric acid(C₁₀).
 12. The method of claim 9, wherein the fish oil is selected fromsardine, salmon, herring, mackerel and/or other cold water fish oils orfish oils synthetically obtainable by reesterification of glycerol withomega-3 fatty acid obtained by hydrolysis of cold water fish oils. 13.The method of claim 9, wherein the fish oil comprises triglycerideswhich contain at least 25% by weight of eicosapentaenoic acid, based onthe total weight of fatty acids of the fish oil.
 14. The method of claim9, wherein fish oil comprises triglycerides which contain at least 12%by weight of docosahexaenoic acid, based on the total weight of fattyacids of the fish oil.
 15. The method of claim 9, wherein the totallipid content is 5 to 30% by weight, based on the total volume of thelipid emulsion.
 16. The method of claim 9, further comprising at leastone auxiliary agent or additive selected from the group consisting ofemulsifiers, emulsifying acids (co-emulsifiers), stabilizers,antioxidants and isotonizing additives.
 17. The method of claim 9,wherein the organs are selected from the group consisting of heart,kidney, brain, liver, lung and fat tissue.
 18. The method of claim 9,wherein the tissues are selected from the group consisting ofendothelium, white blood cells, platelets and immune cells.
 19. Theisotonic lipid emulsion of claim 1, wherein the weight ratio of mediumchain triglycerides (MCT) to fish oil is 8:2.
 20. A preparation forintravenous bolus injection, the preparation comprising an isotoniclipid-in-water emulsion free of long-chain vegetable oils, said emulsioncomprising I. 78 to 95% by weight of medium chain triglycerides (MCT),and II. 5 to 22% by weight of fish oil, based on the total amount byweight of MCT and fish oil lipids in the emulsion, wherein the fish oilhas an eicosapentaenoic acid content from 20 to 40 percent based on thefatty acid methyl ester of the fish oil.
 21. The preparation forintravenous bolus injection of claim 20, wherein the medium chaintriglyceride fatty acids contain between 6 and 14 carbon atoms.
 22. Thepreparation for intravenous bolus injection of claim 21, wherein themedium chain triglycerides are comprised of at least 90% oftriglycerides of caprylic acid (C₈) and capric acid (C₁₀).
 23. Thepreparation for intravenous bolus injection of claim 20, wherein thefish oil is selected from sardine, salmon, herring, mackerel and/orother cold water fish oils or fish oils synthetically obtainable byreesterification of glycerol with omega-3 fatty acid obtained byhydrolysis of cold water fish oils.
 24. The preparation for intravenousbolus injection of claim 20, wherein the fish oil comprisestriglycerides which contain at least 25% by weight of eicosapentaenoicacid, based on the total weight of fatty acids of the fish oil.
 25. Thepreparation for intravenous bolus injection of claim 20, wherein thefish oil comprises triglycerides which contain at least 12% by weight ofdocosahexaenoic acid based on the total weight of fatty acids of thefish oil.
 26. The preparation for intravenous bolus injection of claim20, wherein the total lipid content is 5 to 30% by weight, based on thetotal volume of the lipid emulsion.
 27. The preparation for intravenousbolus injection of claim 20, further comprising at least one auxiliaryagent or additive selected from the group consisting of emulsifiers,emulsifying acids (co-emulsifiers), stabilizers, antioxidants andisotonizing additives.
 28. The preparation for intravenous bolusinjection of claim 20, wherein the weight ratio of medium chaintriglycerides (MCT) to fish oil is 8:2.